elise saunier bionovo, inc. nams 2009 identification of tissue-selective er agonists from plants

Elise Saunier

Bionovo, Inc. NAMS 2009

Identification of Tissue-Selective ERIdentification of Tissue-Selective ER agonists from Plantsagonists from Plants

Endocrine changes during the menopausal transition, rather than the aging Endocrine changes during the menopausal transition, rather than the aging process, are related to changes in body weight and fat distribution.process, are related to changes in body weight and fat distribution.

In age-matched women: higher percentage of fat tissue in peri- and post-In age-matched women: higher percentage of fat tissue in peri- and post-menopausal women (p<0.001) compare to premenopausal and menopausal women (p<0.001) compare to premenopausal and postmenopausal + HRT womenpostmenopausal + HRT women

Perimenopausal and postmenopausal women show a shift to a central, Perimenopausal and postmenopausal women show a shift to a central, android fat distribution. Regional distribution shows higher fat distribution android fat distribution. Regional distribution shows higher fat distribution in the trunk (p<0.001) and lower fat in the legs (p<0.05) for the in the trunk (p<0.001) and lower fat in the legs (p<0.05) for the postmenopausal womenpostmenopausal women

Overweight and obesity are associated with increased morbidity and Overweight and obesity are associated with increased morbidity and mortality, with central distribution of body fat recognized as an independent mortality, with central distribution of body fat recognized as an independent predictor of cardiovascular disease in womenpredictor of cardiovascular disease in women

Can be counteracted by HRTCan be counteracted by HRT

Weight Gain during MenopauseWeight Gain during Menopause

2

Fat redistribution during menopauseFat redistribution during menopause

*p<0.05 (vs. premenopause value); **p<0.01 (vs premenopause value)3

Genazzani & Gambacciani studied the modifications of body weight and fat Genazzani & Gambacciani studied the modifications of body weight and fat distribution from 2529 womendistribution from 2529 women

Mammary GlandMammary GlandUterusUterus

Breast CancerBreast CancerEndometrial CancerEndometrial Cancer

Adipose TissueAdipose Tissue

Decreased ObesityDecreased ObesityDecreased Metabolic SyndromeDecreased Metabolic Syndrome

ER

Estrogen

in HT

Estrogen

in HT

ER

Estrogen

in HT

Estrogens in HT are not Tissue SelectiveEstrogens in HT are not Tissue Selective

4



ER

Tissue Selective ERTissue Selective ERαα-estrogens-estrogens

5

Tissue Selective

ERα Agonist

Tissue Selective

ERα Agonist



ER

Tissue Selective

ERα Agonist

SAFERSAFERSAFERSAFER

To identify tissue selective ERTo identify tissue selective ER agonists in plants agonists in plants

MethodsMethods

1.1. Screened over 50 plant extracts for activation of an ERE reporter Screened over 50 plant extracts for activation of an ERE reporter genegene

2.2. Selected two plant extracts for testing in mice by gavage:Selected two plant extracts for testing in mice by gavage:

#3 from Radix Glycyrrhiza#3 from Radix Glycyrrhiza#39 from Radix Pueraria#39 from Radix Pueraria

3.3. Ovariectomized mice were pre-fed for 50 days with HFD before Ovariectomized mice were pre-fed for 50 days with HFD before started treatment with E2, #3 and #39 for 42 daysstarted treatment with E2, #3 and #39 for 42 days

4.4. Measured weights of body, abdominal fat, MG and uterusMeasured weights of body, abdominal fat, MG and uterus

5.5. Determine gene expression profiles in abdominal fat, MG and Determine gene expression profiles in abdominal fat, MG and uterusuterus

GoalGoal

6

7

Plant extract #3 and #39 induce ERE-Luciferase reporter expression in vitroPlant extract #3 and #39 induce ERE-Luciferase reporter expression in vitro

Plant Extracts have ERα ActivityPlant Extracts have ERα Activity

*p<0.05 vs. control

ERE-ERα tk-Luc

0

1

2

3

4

10-8 0.2 0.5 1 2.5 10 20 100 0.2 0.5 1 2.5 10 20 100

RL

UControlE2#3#39

**

**

***

*****

EC50 = 16 μg/mlEC50 = 16 μg/ml EC50 = 5.5 μg/mlEC50 = 5.5 μg/ml

4.8

5.0

5.2

5.4

5.6

5.8

0.1 1 10 100 1000

concentration (ug/ml)

4.8

5.0

5.2

5.4

5.6

5.8

0.1 1 10 100 1000

concentration (ug/ml)

Binding assay

8

Consumption of a high fat diet increases body weight in miceConsumption of a high fat diet increases body weight in mice

E2 administration reverses the weight gainE2 administration reverses the weight gain

Plant extract #3 and #39 also reverse weight gainPlant extract #3 and #39 also reverse weight gain

Decrease in Body Weight by the Plant ExtractsDecrease in Body Weight by the Plant Extracts

-30

-20

-10

0

10

7 14 21 28 35 42

days in HFD

% w

eig

ht

chan

ge

vehicle

E2

3

39

***

**

*

**

*

**

*

*

*

*

**

*p<0.05 vs. control

Abdominal fat

0

0.5

1

1.5

2

2.5w

eig

ht

(g) vehicle

E2

3

39

* *

*

9

#3 and #39 mimic estradiol’s effect on the abdominal fat#3 and #39 mimic estradiol’s effect on the abdominal fat

Plant Estrogens Decrease Abdominal FatPlant Estrogens Decrease Abdominal Fat

Abdominal FatAbdominal FatAbdominal FatAbdominal Fat

*p<0.05 vs. control

Uterus

0

100

200

300

wei

gh

t (m

g) vehicle

E2

3

39

*

10

Estradiol treatment induces a massive increase in uterus weightEstradiol treatment induces a massive increase in uterus weight

#3 and #39 do not mimic estradiol’s effect on the uterus#3 and #39 do not mimic estradiol’s effect on the uterus

Uterus Weight is not Increased by Plant ExtractsUterus Weight is not Increased by Plant Extracts

UterusUterusUterusUterus

*p<0.05 vs. control

Mammary gland

0

0.5

1

1.5

2

wei

gh

t (g

) vehicle

E2

3

39

**

*

11

Estradiol reduces the mammary gland weight but induces Estradiol reduces the mammary gland weight but induces

development of ducts (elongation, branching)development of ducts (elongation, branching)

Mammary gland is not affected by the Plant Mammary gland is not affected by the Plant ExtractsExtracts

Mammary GlandMammary GlandMammary GlandMammary Gland controlcontrol

3399

33

EE22

While the weight of the mammary gland is diminished by the plant While the weight of the mammary gland is diminished by the plant

extracts, the ducts do not develop.extracts, the ducts do not develop.

*p<0.05 vs. control

12

#3 and #39 mimic estradiol’s effect on the abdominal fat#3 and #39 mimic estradiol’s effect on the abdominal fat

#3 and #39 DO NOT mimic estradiol’s effect on the uterus#3 and #39 DO NOT mimic estradiol’s effect on the uterus

Plant Extracts have Tissue Specific Plant Extracts have Tissue Specific Estrogenic EffectsEstrogenic Effects



Mammary GlandMammary GlandMammary GlandMammary Gland

To understand molecular basis for tissue specific effectsTo understand molecular basis for tissue specific effects

gene expression profile by microarraygene expression profile by microarray

13

Compared to estradiol, #3 and #39 regulate a comparable number of genes in Compared to estradiol, #3 and #39 regulate a comparable number of genes in

the abdominal fatthe abdominal fat

Gene regulation by the Plant Extracts is Tissue Gene regulation by the Plant Extracts is Tissue SpecificSpecific

-800

-400

0

400

800

E2 #3 #39

-400

-200

0

200

400

E2 #3 #39

-1500-1000

-5000

5001000

1500

E2 #3 #39

Mammary GlandMammary GlandMammary GlandMammary GlandUterusUterusUterusUterus


However, little or no genes are induced or repressed in the Uterus and MG, However, little or no genes are induced or repressed in the Uterus and MG,

when compare to E2 treatment.when compare to E2 treatment.

14

847

E2E2

#3#3

239239

4848

413413

338

#39#39

45 349

Mammary GlandMammary GlandMammary GlandMammary Gland

469

00

1012

E2E2 #39#39

#3#3

Tissue Specific Activity of Plant EstrogensTissue Specific Activity of Plant Estrogens

1980

#3#3

8181

11112112

2525

167 304

E2E2#39#39


Abdominal FatAbdominal FatAbdominal FatAbdominal FatE2, #3 and 39 regulate E2, #3 and 39 regulate genes in the fatgenes in the fat

E2 is the major gene E2 is the major gene regulator in the uterus regulator in the uterus

and the mammary glandand the mammary gland

158 Inflammatory 158 Inflammatory genes repressed by genes repressed by E2, #3 and #39E2, #3 and #39





ER

Estrogen

in HT

Estrogen

in HT

ER

Estrogen

in HT

Estrogens in HT are not Tissue SelectiveEstrogens in HT are not Tissue Selective

15

E2E2

669

FatFat

MGMG

7373 99 79

228

UterusUterus

338 1749

modifies gene expression in all modifies gene expression in all

3 tissues3 tissues



ER

Tissue Selective ERTissue Selective ERαα-estrogens-estrogens

16

Tissue Selective

ERα Agonist

Tissue Selective

ERα Agonist #3#3MGMG

190190 848848 3939

00

FatFat UterusUterus

#39#39

734

MGMG

306

66

358

16

FatFat UterusUterus

Plant Estrogens

#3 and #39 have one major target #3 and #39 have one major target abdominal FAT = GOOD NEWSabdominal FAT = GOOD NEWS

Much less or no effect on MG and Much less or no effect on MG and UterusUterus

•Plant extracts mimic the beneficial effect of Plant extracts mimic the beneficial effect of estradiol on body weight and repression of estradiol on body weight and repression of inflammatory genesinflammatory genes

•Plant extracts have little or no effect on the Plant extracts have little or no effect on the mammary gland and uterusmammary gland and uterus

•Animal data support the goal to use these Animal data support the goal to use these plants in clinical trials to safely treat obesity in plants in clinical trials to safely treat obesity in postmenopausal womenpostmenopausal women

•These studies demonstrate that it is possible These studies demonstrate that it is possible to discover tissue selective ERto discover tissue selective ER agonists. Might agonists. Might have a more favorable clinical profile than have a more favorable clinical profile than estrogens used currently in classic HT for estrogens used currently in classic HT for menopausal symptomsmenopausal symptoms

SummarySummary

elise saunier bionovo, inc. nams 2009 identification of tissue-selective er agonists from plants

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